U.S. patent application number 12/074865 was filed with the patent office on 2009-09-10 for glp-1 gene delivery for the treatment of type 2 diabetes.
Invention is credited to Kyungsoo Ko, Minhyung Lee, Seungjoon Oh.
Application Number | 20090227660 12/074865 |
Document ID | / |
Family ID | 29548661 |
Filed Date | 2009-09-10 |
United States Patent
Application |
20090227660 |
Kind Code |
A1 |
Oh; Seungjoon ; et
al. |
September 10, 2009 |
GLP-1 gene delivery for the treatment of type 2 diabetes
Abstract
This patent discloses compositions and methods of use thereof to
normalize the blood glucose levels of patients with type 2
diabetes. It relates particularly to a plasmid comprising a chicken
.beta. actin promoter and enhancer; a modified GLP-1 (7-37) cDNA
(p.beta.GLP1), carrying a furin cleavage site, which is constructed
and delivered into a cell for the expression of active GLP-1.
Inventors: |
Oh; Seungjoon; (Seoul,
KR) ; Lee; Minhyung; (Seoul, KR) ; Ko;
Kyungsoo; (Seoul, KR) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE, 32ND FLOOR
CHICAGO
IL
60606
US
|
Family ID: |
29548661 |
Appl. No.: |
12/074865 |
Filed: |
March 5, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10153470 |
May 21, 2002 |
7374930 |
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12074865 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 47/593 20170801;
A61K 48/00 20130101; A61K 47/59 20170801; C07K 14/605 20130101;
A61K 38/00 20130101; A61K 47/645 20170801 |
Class at
Publication: |
514/44.R |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1. A method of normalizing blood glucose levels of a warm blooded
animal having type 2 diabetes, comprising: providing a transfection
formulation comprising a cationic polymeric gene carrier complexed
with a selected plasmid consisting essentially of: an expression
facilitating sequence derived from chicken .beta.-actin promoter
and enhancer; an expression sequence comprising ATG (start codon)
followed by a sequence SEQ ID NO:5 (CGTCAACGTCGT) coding for a
furin cleavage site (FCS) and a sequence coding for the active form
of GLP-1 (7-37) or derivatives thereof that are operably linked to
said expression facilitating sequence in a proper charge ratio
(positive charge of the copolymer/negative charge of the nucleic
acid) that is optimally effective for both in vivo and in vitro
transfection; and administering to the animal to be treated an
effective amount of the composition such that the cell internalizes
the GLP-1 gene.
2. The method of claim 1, wherein the cationic polymeric gene
carrier is PAGA.
3. The method of claim 2, wherein the weight ratio of DNA to PAGA
is preferably within a range of 1:0.82 to 1:2.46.
4. The method of claim 2, wherein plasmid comprising an nucleotide
sequence represented by the SEQ ID NO:1 or having the nucleotide
sequence represented by the SEQ ID NO:2.
5. The method of claim 2, wherein the cationic polymeric gene
carrier further comprises a targeting moiety.
Description
PRIORITY DATA
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/153,470, filed May 21, 2002, which is incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to compositions and methods of use
thereof to normalize the blood glucose levels of patients with type
2 diabetes. More particularly, the invention relates to a
composition and method for the delivery of the GLP-1 gene, both in
vitro and in vivo, for the treatment of type 2 diabetes. It relates
particularly to a plasmid comprising a chicken .beta. actin
promoter and enhancer; a modified GLP-1 (7-37) cDNA (p.beta.GLP1),
carrying a furin cleavage site, which is constructed and delivered
into a cell for the expression of active GLP-1. The invention also
encompasses transfecting compositions comprising a complex of a
plasmid containing a modified GLP-1 (7-37) cDNA (p.beta.GLP1) and
poly(ethylenimine) (PEI) (for in vitro gene delivery) or PAGA (for
in vivo gene delivery).
BACKGROUND OF INVENTION
[0003] Type 2 diabetes is characterized by hyperglycemia, insulin
resistance, absolute or relative insulin deficiency,
hyperglucagonemia, and increased hepatic glucose production.
Although many treatment trials for type 2 diabetes have been held,
there is still no definitive treatment for the disease. Insulin
secretion is modulated by incretin hormones which are produced by
the intestinal enteroendocrine cells and constitute one arm of the
enteroinsular axis. There are two major incretins. One is
glucose-dependent insulinotrophic polypepetide (GIP) and the other
is glucagon like peptide-1 (GLP-1). These two incretin hormones
account for 20% and 80% respectively, of the intestinal incretin
effect. Holst J J: Glucagonlike peptide 1: a newly discovered
gastrointestinal hormone. Gastroenterology 107:1848-1855, 1994 GIP,
but not GLP-1, tends to lose its actions in patients with type 2
diabetes. Nauck M A, Heimesaat M M, Orskov C, Holst J J, Ebert R,
Creutzfeldt W: Preserved incretin activity of glucagons-like
peptide 1 [7-36 amide] but not of synthetic human gastric
inhibitory polypeptide in patients with type-2 diabetes mellitus. J
Clin Invest 91:301-307, 1993. GLP-1 was recently used for the
treatment of type 2 diabetes. See U.S. Pat. Nos. 5,614,492;
5,545,618 and 6,048,724 which are incorporated herein by
reference.
[0004] GLP-1, produced by intestinal L-cells, stimulates glucose
induced insulin secretion and inhibits glucagon secretion. GLP-1
has two active forms, GLP-1 (7-36) amide and GLP-1 (7-37), that are
products of posttranslational processing of proglucagon in
mammalian intestinal cells. The active forms of GLP-1 are
degradable in the plasma by the action of dipeptidyl peptidase IV.
During degradation, the active form of GLP1 (7-36 or 7-37) loses
its N-terminal amino acid residues and results in an inactive form
of GLP-1 (9-36 amide). Therefore, the active forms of GLP-1 have
very short plasma half lives (about 5 minutes) and metabolic
clearance rates. Fehmann H C et. al: Endocr Rev 16:390-410, 1995
There have been several studies on administration of GLP-1 to type
2 diabetic patients which have shown that GLP-1 effectively reduces
hyperglycemia in type 2 diabetic patients. Nauck et. al. J Clin
Invest 91:301-307, 1993; Nauck et. Al: Diabetes Care 21:1925-1931,
1998; and Rachman J et. al Diabetologia 40:205-211, 1997. However,
it is very difficult to consistently deliver the active form of
GLP-1 because of its short half-life. Even when using the long
acting form of GLP-1, exendin-4, twice daily administration is
required to maintain a normal glucose level. Szayna M, Doyle M E,
Betkey J A, Holloway H W, Spencer R G, Greig N H, Egan J M:
Exendin-4 decelerates food intake, weight gain, and fat deposition
in Zucker rats. Endocrinology 141:1936-1941, 2000
[0005] Gene therapy is generally considered as a promising
approach, not only for the treatment of diseases with genetic
defects, but also in the development of strategies for treatment
and prevention of chronic diseases such as cancer, cardiovascular
disease and diabetes. However, nucleic acids, as well as other
polyanionic substances are rapidly degraded by nucleases and
exhibit poor cellular uptake when delivered in aqueous solutions.
Since early efforts to identify methods for delivery of nucleic
acids in tissue culture cells in the mid 1950's, steady progress
has been made towards improving delivery of functional DNA, RNA,
and antisense oligonucleotides in vitro and in vivo.
[0006] The gene carriers used so far include viral systems
(retroviruses, adenoviruses, adeno-associated viruses, or herpes
simplex viruses) or nonviral systems (liposomes, polymers,
peptides, calcium phosphate precipitation and electroporation).
Viral vectors have been shown to have high transfection efficiency
when compared to non-viral vectors, but due to several drawbacks,
such as targeting only dividing cells, random DNA insertion, their
low capacity for carrying large sized therapeutic genes, risk of
replication, and possible host immune reaction, their use in vivo
is severely limited.
[0007] An ideal transfection reagent should exhibit a high level of
transfection activity without the need for any mechanical or
physical manipulation of cells or tissues. The reagent should be
non-toxic, or minimally toxic, at the effective dose. It should
also be biodegradable in order to avoid any long term adverse side
effects on the treated cells. When gene carriers are used for
delivery of nucleic acids in vivo, it is essential that the gene
carriers themselves be nontoxic and that they degrade into
non-toxic products. To minimize the toxicity of the intact gene
carrier and its degradation products, the design of gene carriers
needs to be based on naturally occurring metabolites.
[0008] As compared to viral gene carriers, there are several
advantages to the use of non-viral based gene therapies, including
their relative safety and low cost of manufacture. There are
several polymeric materials currently being investigated for use as
gene carriers, of which poly-L-lysine (PLL) is the most popular,
but few of them are biodegradable. In general, polycationic
polymers are known to be toxic and the PLL backbone is barely
degraded under physiological conditions. It remains in cells and
tissues and causes an undesirably high toxicity. In addition, like
most cationic polymers, PLL/DNA complexes have drawbacks including
precipitation as insoluble particles and the tendency to aggregate
into larger complexes under physiological conditions.
[0009] In view of the foregoing, there is a need for the
development of a composition and a gene therapy method for the
treatment of type-2 diabetes wherein the gene carrier is soluble
and biodegradable, meaning that the non-viral polymer gene carrier
can break down or degrade within the body to non-toxic components
after the genes have been delivered.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention provides a composition and a method
for delivery of the GLP-1 gene, both in vitro and in vivo, for the
treatment of type 2 diabetes. Particularly, the present invention
provides a plasmid comprising a chicken .beta. actin promoter and
enhancer; a modified GLP-1 (7-37) cDNA (p.beta.GLP1) carrying a
furin cleavage site. The invention also encompasses transfecting
compositions comprising a complex of a plasmid containing a
modified GLP-1 (7-37) cDNA (p.beta.GLP1) and poly(ethylenimine)
(PEI) (for in vitro gene delivery) or PAGA (for in vivo gene
delivery), at a proper weight ratio. Particularly, the weight ratio
of DNA to the cationic biodegradable gene carrier is preferably
within a range 1:0.85 to 1:2.90. For DNA/PAGA, the ratio is
preferably with the range of 1:0.82 to 1:2.46 and for DNA/PEI, the
ratio is preferably with the range of 1:0.676 to 1:2.704 for
(DNA/PEI).
[0011] This invention also provides for a method of transfecting a
cell with the GLP-1 gene comprising the steps of:
[0012] (a) providing a composition comprising effective amounts of
complexes of a plasmid containing a modified GLP-1 (7-37) cDNA
(p.beta.GLP1) and a polycationic gene carrier at a proper weight
ratio;
[0013] (b) contacting the cell with an effective amount of the
composition such that the cell internalizes the GLP-1 gene; and
[0014] (c) culturing the cell with the internalized DNA under
conditions favorable for the growth thereof.
[0015] This invention further provides for a method of normalizing
the blood glucose levels of an animal with type 2 diabetes,
comprising the steps of:
[0016] (a) providing a composition comprising effective amounts of
complexes of a plasmid containing a modified GLP-1 (7-37) cDNA
(p.beta.GLP1) and poly[.alpha.-(4-aminobutyl)-L-glycolic acid]
(PAGA) at a proper charge ratio,
[0017] (b) administering to the animal to be treated an effective
amount of the composition such that the cell internalizes the GLP-1
gene.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic representation of plasmid p.beta.GLP1
carrying a modified GLP-1 (7-37) cDNA sequence and a furin cleavage
site.
[0019] FIG. 2 shows the RT-PCR assay of the production of GLP-1 in
HepG2 cells transfected by the transfection composition containing
p.beta.GLP1/gene carrier complexes.
[0020] FIG. 3 shows the insulin secretion of co-cultured islets
with HepG2 cells transfected by the transfection composition
containing p.beta.GLP1/gene carrier complexes.
[0021] FIG. 4. illustrates the therapeutic effect of the
transfection composition containing p.beta.GLP1/PAGA complexes in
Zucker Diabetic Fatty (ZDF) rats.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Before the present composition and method for treatment of
type-2 diabetes are disclosed and described, it is to be understood
that this invention is not limited to the particular
configurations, process steps, and materials disclosed herein as
such configurations, process steps, and materials may vary
somewhat. It is also to be understood that the terminology employed
herein is used for the purpose of describing particular embodiments
only and is not intended to be limiting since the scope of the
present invention will be limited only by the appended claims and
equivalents thereof.
[0023] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise. In
describing and claiming the present invention, the following
terminology will be used in accordance with the definitions set
forth below.
[0024] "Transfecting" or "transfection" shall mean transport of
nucleic acids from the environment external to a cell to the
internal cellular environment, with particular reference to the
cytoplasm and/or cell nucleus. Without being bound by any
particular theory, it is understood that nucleic acids may be
delivered to cells either after being encapsulated within or
adhering to one or more cationic lipid/nucleic acid complexes or
entrained therewith. Particular transfecting instances deliver a
nucleic acid to a cell nucleus. Nucleic acids include both DNA and
RNA as well as synthetic congeners thereof. Such nucleic acids
include as well as protein producing nucleotides, on and off and
rate regulatory nucleotides that control protein, peptide, and
nucleic acid production. In particular, but nonlimiting, they can
be genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or
synthetic or semi-synthetic sequences, and of natural or artificial
origin. In addition, the nucleic acid can be variable in size,
ranging from oligonucleotides to chromosomes. They may be obtained
by any technique known to a person skilled in the art.
[0025] As used herein, the term "biodegradable" or "biodegradation"
is defined as the conversion of materials into less complex
intermediates or end products by solubilization hydrolysis, or by
the action of biologically formed entities which can be enzymes and
other products of the organism.
[0026] As used herein, "effective amount" means an amount of a
nucleic acid or bioactive agent that is sufficient to provide the
desired local or systemic effect and performance at a reasonable
risk/benefit ratio as would attend any medical treatment.
[0027] As used herein, "administering", and similar terms means
delivering the composition to the individual being treated such
that the composition is capable of being circulated systemically to
where the composition binds to a target cell and is taken up by
endocytosis. Thus, the composition is preferably administered to
the individual systemically, typically by subcutaneous,
intramuscular, intravenous, or intraperitoneal administration.
Injectables for such use can be prepared in conventional forms,
either as a liquid solution or suspension, that is suitable for
preparation as a solution or suspension in a liquid prior to
injection, or as an emulsion. Suitable excipients that can be used
for administration include, for example, water, saline, dextrose,
glycerol, ethanol, and the like; and if desired, minor amounts of
auxiliary substances such as wetting or emulsifying agents,
buffers, and the like.
[0028] According to the invention there is provided a plasmid
suitable for GLP-1 eukaryotic expression, consisting essentially
of: an expression facilitating sequence derived from chicken
.beta.-actin promoter and enhancer; an expression sequence
comprising ATG (start codon) followed by a sequence (CGTCAACGTCGT)
coding for a furin cleavage site (FCT) and a sequence coding for
the active form of GLP-1 (7-37) that are operably linked to said
expression facilitating sequence. Optionally, the plasmid may
contain a non-mammalian origin of replication; and a sequence
operably encoding a selectable marker.
[0029] One important aspect of the present invention relates to the
addition of the furin cleavage site between the start codon and the
GLP-1 (7-37) gene. It is common knowledge that all gene expression
requires a start codon which encodes methionine at the N terminal
of a protein. Many active peptides such as GLP-1 are products of
post-translation processes in vivo. If the N terminal amino acid of
GLP-1 (methionine) produced from the plamid is not cleaved, it will
block the activity of the first two amino acids of GLP-1 which are
the receptor binding domain, which in turn makes the GLP-1 lose its
biological activity completely. In order to construct a plasmid
that can express high levels of active forms of GLP-1, a furin
cleavage site (FCT) is introduced between the start codon and the
GLP-1 (7-37) sequence. Furin is a type I transmembrane protein
composed of a signal peptide, a propeptide terminating in an
endoproteolytic cleavage site comprised of a cluster of basic
residues, a subtilisin-like catalytic domain, a middle domain, and
a cysteine-rich domain followed by C-terminal transmembrane anchor
and a cytosolic tail. Therefore, when the GLP-1 produced from the
plasmid constructs, according to the present invention, are
secreted from the cells, the N terminal methionine-FCT of the
expression product will be cleaved by the furin in the Golgi
apparatus of the cells. One benefit of choosing FCT is that furin
is widely distributed in most cell types, so that the plasmid
constructs of the present invention can efficiently express active
GLP-1 in almost any type of cell in the body. Although the plasmid
constructs of the present invention are disclosed for in vivo gene
delivery of active GLP-1, the usage of FCT is applicable to gene
delivery of any peptide whose activity requires cleavage of the
N-terminal methionine of the expression product and they intended
to be within the scope of the invention.
[0030] In one embodiment of the present invention, the plasmid may
possess the functional characteristics of the plasmid comprising a
nucleotide sequence represented by SEQ ID NO:1. One example of the
plasmid of the present invention has a nucleotide sequence
represented by SEQ ID NO:2.
[0031] In another embodiment, the plasmid consisting essentially
of: an expression facilitating sequence derived from chicken
.beta.-actin promoter and enhancer; an expression sequence
comprising ATG (start codon) followed by a sequence (CGTCAACGTCGT)
coding for a furin cleavage site (FCT) and a sequence coding for
the active form of GLP-1 (7-37) or derivatives thereof that are
operably linked to said expression facilitating sequence.
[0032] In another embodiment, the invention provides a host cell
transformed by any of the above plasmids.
[0033] In still another embodiment, the invention provides a method
for producing any of the above plasmids, comprising the steps of:
growing bacterial cells containing the plasmid; and recovering the
plasmid from the bacterial cells.
[0034] In an additional embodiment, the invention provides a
pharmaceutical composition comprising the plasmid in combination
with a pharmaceutically acceptable vehicle. The plasmid may be
complexed to a biodegradable cationic polymeric gene carrier
[0035] The invention also provides a eukaryotic expression vector
for the expression of a DNA sequence in a human tissue, consisting
essentially of: an expression facilitating sequence derived from
chicken .beta.-actin promoter and enhancer; an expression sequence
comprising ATG (start codon) followed by a sequence (CGTCAACGTCGT)
coding for a furin cleavage site (FCT) and a sequence coding for
the active form of a bioactive peptide that are operably linked to
said expression facilitating sequence, and a sequence containing a
transcriptional termination and a polyadenylation signal; and a
selectable marker; wherein the vector is capable of replicating in
prokaryotes.
[0036] In another embodiment, the invention provides a method of
gene therapy, the improvement comprising administering any of the
above plasmids directly into a cell resulting in the local
secretion of active GLP-1, active GLP-1 derivatives or other
peptides.
[0037] It will be readily apparent to one skilled in the art that
various substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0038] This approach differs from previous methods in which cells
are collected, propagated in vitro, modified and selected and then
re-injected in vivo. Limitations of these latter approaches
include, 1) the need to establish a cell line from each
experimental subject to avoid tissue rejection, 2) concerns about
alteration of the phenotype of cells propagated in tissue culture,
3) outgrowth of aberrant transformed cells, and 4) the time and
effort required. In the present method, genes are directly
transferred into an animal with type-2 diabetes where the cells
take up and express the gene. DNA expression is facilitated by
introducing the DNA complexed with a cationic biodegradable gene
carrier. The gene carrier facilitates entry of the DNA into those
cells thus providing for intracellular access to the DNA/gene
carrier complex. Delivery of DNA to patients in a drug-like manner
is thus facilitated.
[0039] Classical pharmacological studies of drug distribution, half
life, metabolism, and excretion are not entirely relevant to in
vivo gene injection and expression. However, the fate of the
plasmid and detection of the gene product (GLP-1) are relevant to
the development of this agent. Therefore, as part of the
measurement of the efficacy of this study, successful gene transfer
and expression is evaluated by molecular and immunological
analyses. The following parameters are measured to evaluate the
transfection and expression of active GLP-1 (7-37): 1) the presence
of mRNA which encodes the Met-FCS-GLP-1 from the plasmid is
assessed by RT-PCR amplification of cells obtained after
transfection, 2) secretion of active GLP-1 from the cells
transfected with the plasmid in vitro is measured by enzyme linked
immunosorbent assay (ELISA), 3) measurement of insulin secretion of
co-cultured islets with GLP-1 plamid transfected cells, and 4)
serum glucose levels are measured pre-treatment and after the start
of therapy.
[0040] The plasmid suitable for GLP-1 expression is a eukaryotic
expression vector that codes for Met-FCS-GLP-1 (7-37). A process
for the production of this plasmid has been developed using E. coli
(JM109). The process is scaleable and is a combination of highly
reproducible unit operations (fermentation, cell lysis,
precipitation, size exclusion chromatography, formulation and
fill). The advantages over existing methods include scalability,
improved plasmid purity and the elimination of undesirable process
additives such as toxic solvents and animal derived enzymes. One
skilled in the art will readily appreciate that the GLP-1 plasmid
described herein is representative of a preferred embodiment that
is exemplary and not intended to be a limitation on the scope of
the invention
[0041] Any eukaryotic expression vector that is adapted to carry
out the objectives and obtain the ends and advantages mentioned as
well as those inherent herein is encompassed within the spirit of
the invention. Accordingly, the plasmids of the present invention
are assembled out of components comprising a gene encoding an
active peptide such as GLP-1, origins, promoters, polyadenylation
signals, and furin cleavage sites (FCS).
[0042] The p.beta.GLP-1 plasmid of the present invention is
diagrammed in FIG. 1, and the nucleotide sequence of the coding
strand of the plasmid is given as SEQ ID NO:2. This is a high copy
number plasmid that was constructed using isolated segments of
synthesized cDNA of GLP-1 (3-37, using standard molecular genetic
techniques and commercially available enzymes. The backbone plasmid
DNA is derived from pCI (Promega Inc.), a commercially available
vector widely used in molecular biology laboratories.
[0043] Development of a safe and efficient gene delivery carrier is
an important factor to the success of gene therapy. Preferably, the
cationic biodegradable polymeric gene carrier used in the present
invention can spontaneously form discrete nanometer-sized particles
with a nucleic acid, which can promote more efficient gene
transfection into mammalian cells and show reduced cell toxicity.
The biodegradable gene carrier, such as PAGA, is readily
susceptible to metabolic degradation after incorporation into
animal cells. Moreover, the polymeric gene carrier can form an
aqueous micellar solution which is particularly useful for the
systemic delivery of genes. PAGA is disclosed in U.S. Pat. No.
6,217,912, hereby fully incorporated by reference.
[0044] The present invention further provides transfection
formulations, comprising a biodegradable cationic polymeric gene
carrier complexed with an expressive gene vector carrying the GLP-1
gene, in the proper weight ratio (positive charge of the gene
carrier/negative charge of the nucleic acid), that is optimally
effective for both in vivo and in vitro transfection. Preferably,
the biodegradable gene carrier is PAGA and the gene vector is
p.beta.GLP1. Particularly, the weight ratio of DNA to the cationic
biodegradable gene carrier is preferably within a range 1:0.85 to
1:2.90. For DNA/PAGA, the ratio is preferably with the range of
1:0.82 to 1:2.46 and for DNA/PEI, the ratio is preferably with the
range of 1:0.676 to 1:2.704 for (DNA/PEI).
[0045] The gene carrier of the present invention can also be
conjugated, either directly or via spacer molecules, with targeting
ligands. The targeting ligands conjugated to the gene carrier
direct the gene carrier-nucleic acid/drug complex to bind to
specific target cells and penetrate into such cells. The targeting
ligand can also be an intracellular targeting element, enabling the
transfer of the nucleic acid/drug to be guided towards certain
favored cellular compartments (mitochondria, nucleus, and the
like).
[0046] An advantage of the present invention is that it provides an
efficient transfecting composition for delivery of a GLP-1 gene
into a cell wherein the particle size and charge density are easily
controlled. Control of particle size is crucial for optimization of
a gene delivery system because the particle size often governs the
transfection efficiency, cytotoxicity, and tissue targeting in
vivo. In general, in order to enable its effective penetration into
tissue, the size of a gene delivery particle should not exceed the
size of a virus. In a preferred embodiment of the invention, the
particle sizes will range from about 80 to 200 nm depending on the
cationic copolymer composition and the mixing ratio of the
components. It is known that particles, nanospheres, and
microspheres of different sizes, when injected, accumulate in
different organs of the body depending on the size of the particles
injected. For example, after systemic administration, particles of
less than 150 nm diameter can pass through the sinusoidal
fenestrations of the liver endothelium and become localized in the
spleen, and bone marrow. Intravenous, intra-arterial, or
intraperitoneal injection of particles approximately 0.1 to 2.0
.mu.m diameter leads to rapid clearance of the particles from the
blood stream by macrophages of the reticuloendothelial system.
[0047] It is believed that the presently claimed composition is
effective in delivering the GLP-1 gene by endocytosis. The cationic
polymeric gene carrier may further comprises a targeting moiety is
selected from the group consisting of transferring,
asialoglycoprotein, antibodies, antibody fragments, low density
lipoproteins, interleukins, GM-CSF, G-CSF, M-CSF, stem cell
factors, erythropoietin, epidermal growth factor (EGF), insulin,
asialoorosomucoid, mannose-6-phosphate, mannose, Lewis.sup.x and
sialyl Lewis.sup.x, N-acetyllactosamine, galactose, lactose, and
thrombomodulin, fusogenic agents such as polymixin B and
hemaglutinin HA2, lysosomotrophic agents, and nucleus localization
signals (NLS). Nucleic acid transfer to targeted cells can be
carried out by matching a cell having a selected receptor thereof
with a selected targeting moiety. The present invention therefore
provides methods for treating type 2 diabetes by in vivo delivery
of an expressive gene vector which provides a desirable amount of
GLP-1 protein during a sustained period of time.
[0048] The biodegradable transfecting composition of the present
invention, as described herein, exhibits improved cellular binding
and uptake characteristics toward the GLP-1 gene to be delivered.
As such, the present invention overcomes the problems as set forth
above. For example, the biodegradable gene carrier of the present
invention, such as PAGA, is easily hydrolyzed or degraded into low
molecular weight components which will be easily eliminated from
the body. In addition, the degradation products are small,
non-toxic molecules that are subject to renal excretion and are
inert during the period required for gene expression. Moreover,
cells transfected with the plasmid of the present invention,
namely, p.beta.GLP-1 (3-37), can produce the most potent form of
natural GLP-1 peptide. There are two characteristics in the plasmid
constructs of the present invention that afford the capability of
producing the most potent form of natural GLP-1 peptide. First of
all, there is a furin cleavage site between the start codon and the
GLP-1 gene, thus furin cleaves the first amino acid, methionine,
which is encoded by the start codon. Therefore, the cleavage of
methionine preserved the function of the next two amino acids which
is the GLP-1 receptor binding domain and which is essential for
GLP-1 activity. Secondly, the replacement of chicken .beta. actin
promotor of the CMV promotor provides the plamids the capability of
expressing higher amounts of the active GLP-peptide in cells
because chicken .beta. actin promotor is a more potent promotor
than the CMV promotor.
[0049] The following examples will enable those skilled in the art
to more clearly understand how to practice the present invention.
It is to be understood that, while the invention has been described
in conjunction with the preferred specific embodiments thereof,
that which follows is intended to illustrate and not limit the
scope of the invention. Other aspects of the invention will be
apparent to those skilled in the art to which the invention
pertains.
Example 1
Construction of Plasmid Vector
[0050] This example illustrates the construction of the plasmid
which expresses GLP-1 (FIG. 1.). In this plasmid, GLP-1 expression
was driven by chicken .beta. actin promoter and enhancer. GLP-1
(7-37) gene was synthesized using a DNA synthesizer. The start
codon encodes methionine. If the N-terminal amino acid of the
produced GLP-1 is methionine, it will not be an active form,
because the first two amino acids of GLP-1 are the receptor binding
domain. A furin recognition site (ArgGlyArgArg: CGTCAACGTCGT) was
inserted into the synthesized GLP-1 (7-37) gene just after the
start codon. When the produced GLP-1 is secreted from the cell, the
methionine is cleaved by the furin in the Golgi apparatus. To
replace the promoter part (chicken .beta. actin promoter and CMV
promoter), chicken .beta.-actin promoter and enhancer were isolated
from pEBActNII by digestion with BglII and XbaI. The pEBActNII was
provided by Dr. Kaneda (Osaka University). The CMV promoter of pCI
(Promega Inc.) was also disgested with BglII and XbaI. Both
digested DNA fragments were confirmed by their size using a gel
electrophoresis. And then the DNA fragments in agarose gel were
extracted using Gel Extraction Kit (Quiagen Inc.) The gel
extraction was performed following their prescribed procedure. The
two extracted fragments were ligated using T4 DNA ligsae (Promega
Inc.) under 17.degree. C. for 16 hours, and we named our final
plasmid p.beta.. The synthesized GLP-1 (7-37) was treated with KpnI
and XbaI and inserted into the KpnI and XbaI sites of p.beta.
plasmid. The gel extraction and ligation methods are same as
described above. The resulting p.beta.GLP1 was amplified in E. coli
JM109 (Promega) and purified by an alkaline lysis method using the
Maxi Prep Kit (Qiagen). The purity of the plasmid was confirmed by
absorbance at 260 nm and 280 nm and the quantity was determined by
absorbance at 260 nm. The constructed plasmid was confirmed by DNA
sequencing.
Example 2
Synthesis of PAGA and Formation of PAGA/DNA Complex
[0051] This example illustrates the preparation of a gene delivery
composition, according to the present invention.
[0052] PAGA was synthesized as previously described in U.S. Pat.
No. 6,217,912, hereby incorporated by reference. Briefly,
CBZ-L-oxylysine was synthesized from CBZ-L-lysine. CBZ-L-oxylysine
was polymerized by a melting condensation reaction at 150.degree.
C. under a vacuum of 10.sup.-4 Torr for 5 days. The polymer was
dissolved with chloroform and precipitated with methanol. The dried
polymer was dissolved with DMF containing a palladium activated
carbon catalyst. 85% formic acid was added as a proton donor. After
15 hours at room temperature, the polymer solution was isolated
from the catalyst by addition of 2 N HCl and the polymer was
precipitated with acetone. PAGA was stored at -70.degree. C. until
use.
[0053] The DNA/carrier complexes were prepared by self assembly.
PEI (25,000 Da) and PAGA were dissolved in PBS (pH 7.3). Ten times
diluted carrier solution was slowly added into the prepared DNA
plasmid and left for 30 minutes to allow for formation of
complexes. The formation of the complexes p.beta.GLP1/PEI and
p.beta.GLP1/PAGA was routinely monitored by 1.0% agarose gel
electrophoresis.
[0054] Stable complexes were formed with PAGA and the aqueous
plasmid DNA solution based on the fact that no precipitation or
aggregation was observed at wide concentration ranges of the
complexes in the PBS buffer. Complex formation of the plasmid DNA
and the cationic copolymer was tested by agarose gel. The results
show that complete neutralization was achieved from the weight
ratio of DNA to the cationic polymer is within a range of 1:0.82 to
1:2.46.
Example 3
Expression of GLP-1 mRNA in Transfected HepG2 Cells
[0055] This example illustrates the expression of GLP-1 mRNA in
transfected HepG2 cells. To analyze the expression of the gene,
RT-PCR was carried out on the p.beta.GLP1/PEI complex that was
transfected into HepG2 cells used to evaluate the expression of
GLP-1.
[0056] HepG2 cells were maintained in MEM supplemented with 10% FBS
in a 5% CO.sub.2 incubator. For the transfection studies, the cells
were seeded at a density of 2.25.times.10.sup.5 cells/6 well
culture plates and incubated for 24 hours before the addition of
the p.beta.GLP1/PEI complex. The p.beta.GLP1/PEI complexes were
prepared by mixing plasmid and PEI at an N/P ratio of 1:5 (the
weight ratio was 1:0.676) in serum free MEM medium and then
incubated for 30 minutes at room temperature. The cells were washed
twice with PBS, and then 2 mL of fresh serum-free MEM media was
added. The p.beta.GLP1/PEI complex was added to each well. The
cells were incubated for 4 hours at 37.degree. C. in a 5% CO.sub.2
incubator. After 4 hours, the transfection mixtures were removed
and 2 mL of fresh MEM containing FBS was added. The cells were
incubated for an additional 24 hrs at 37.degree. C. We collected
the media that was treated with dipeptidyl peptidase IV inhibitor.
Then a GLP-1 assay (Linco Inc.) was carried out following the
suppliers manual
[0057] The transfected cells were washed twice with PBS, and total
RNA was harvested by acid-guanidium thiocyanate-phenol-chloroform
extraction, using RNAwiz (Ambion, Austin, Tex.). The concentration
of RNA was measured by the absorbance at 260 nm, and the integrity
of RNA was confirmed by formaldehyde-formamide denatured agarose
gel electrophoresis. Two micrograms of total RNAs were hybridized
to the backward primer and reverse transcribed using AMV reverse
transcriptase (Promega, Madion, Wis.). The reverse transcribed
samples were amplified by polymerase chain reaction (PCR), using
Taq polymerase (Promega, Madison, Wis.). The sequences of the
specific oligonucleotide primers were as follows: GLP-1 forward
primer, 5'-CAGAAGTTGGTCGTGAGGCA-3'; GLP-1 backward primer,
5'-GCCTTTCACCAGCCAAGCAA-3'. The PCR reaction was performed in 25
cycles at 94.degree. C. for 30 seconds, 55.degree. C. for 30
seconds, 72.degree. C. for 1 minute, followed by an extension of 10
minutes at 72.degree. C. The PCR products were analyzed in 2%
agrose gel electrophoresis. The sizes of the expected products were
110 bp for GLP-1.
[0058] As a result, the GLP-1 mRNA was detected in the p.beta.GLP1
plasmid transfected cells. (FIG. 2A). The transfected HepG2 cells
expressed GLP-1 mRNA in a dose dependent manner. Therefore, this
result suggests that the detected mRNA was expressed by the
exogenous transferred p.beta.GLP1 plasmid, not by the endogenous
GLP-1 gene. Also, the expression level can be controlled by the
dose of p.beta.GLP1 administration.
Example 4
ELISA Detection of GLP-1 in Transfected HepG2 Cells
[0059] This example illustrates the expression of GLP-1 in
transfected HepG2 cells. To detect the expression of the active
GLP-1 peptide, ELISA was carried out on the p.beta.GLP1/PAGA
complex that was transfected into HepG2 cells used to evaluate the
production of active GLP-1.
[0060] HepG2 cells were cultured and transfected with the plamid of
the present invention as described in Example 3. In order to
confirm that the expressed GLP-1 should be secreted and active for
therapeutic purpose, ELISA assays for the active form of GLP-1 were
performed 48 hours after transfection, using the active GLP-1 ELISA
kid (Linco, Inc). As illustrated in FIG. 2B, the active form of
GLP-1 was detected in the cell culture media by an ELISA assay,
suggesting that the expressed GLP-1 by p.beta.GLP1 was secreted
into the cell culture media. The GLP-1 production of the
transfected HepG2 cells was 24.5.+-.0.5 pM/24 hrs (2 .mu.g of
p.beta.GLP1 transfected) and 61.3.+-.6.2 pM/24 hrs (4 .mu.g of
p.beta.GLP1 transfected). However, there was no production of GLP-1
from the negative control.
Example 5
Co-Culture Rat Islets with p.beta.GLP1 Transfected HepG2 Cells
[0061] The example illustrates the effect of the secreted GLP-1 on
the secretion of insulin from rat islets.
[0062] Islets of Langerhans were isolated from male Sprague-Dowley
rat pancreas, by a collagenase digestion technique and
discontinuous Ficoll density gradient centrifugation. Briefly, the
pancreas was removed after swelling caused by collagenase solution
injection (Type V, Sigma, 10-15 ml/pancreas, 1 mg/ml in HBSS)
through the common bile duct, and incubated for 15 min at
37.degree. C. The digested fragments of the pancreas were collected
and washed with HBSS. Finally, the acinar cells and islets were
separated by Ficoll density gradient centrifugation (11%, 20%, 23%,
and 27% in HBSS). The islet rich layer was collected and washed
with HBSS, and the islets were suspended in RPMI-1640 medium with
10% FBS and incubated at 37.degree. C. under humidified conditions
with 5% CO.sub.2. On average, 500-700 islets were isolated from a
rat pancreas. For the co-culture study, the isolated islets were
sub-cultured for 24-48 hours.
[0063] For transfection, HepG2 cells cultured in MEM supplemented
with 10% FBS were used. The cells were seeded at a density of
5.times.10.sup.5/well in a 6 well plate with 1.5 mL medium. After
24 hours incubation, the culture medium was changed to FBS free
fresh medium and loaded with p.beta.GLP1/PEI complexes (PEI/DNA N/P
ratio 5 the weight ration of DNA/PEI is 1:0.676, 4 .mu.g DNA/well)
followed by 4 hours incubation at 37.degree. C. with 5% CO.sub.2.
Then, the transfection medium was changed to fresh MEM with 10% FBS
and the cells were incubated for another 48 hours before the
co-culture study at 37.degree. C. with 5% CO.sub.2.
[0064] The co-culture study was performed with isolated islets and
p.beta.GLP1 transfected HepG2 cells. 30 islets with a small size
distribution (mean size of 150 .mu.m) were carefully transferred
into the p.beta.GLP1 transfected HepG2 cells culture system. The
islets and HepG2 cells were separated by a physical barrier (cell
culture insert). Then, the culture medium was changed to fresh
RPMI-1640 medium (1.5 mL) supplemented with 10% FBS with basal and
high glucose content (50 mg/dL, 300 mg/dL). After 4 h
co-incubation, the insulin and GLP-1 content in the medium was
measured by RIA and ELISA, respectively.
[0065] The transfected HepG2 cells of each well produced from 3.3
pM/4 hrs to 4.6 pM/4 hrs. There was no enhancement of insulin
secretion under a low glucose concentration (50 mg/dL). However, a
remarkable increment of insulin secretion occurred under high
glucose concentrations (300 mg/dL). (FIG. 3.)
[0066] The stimulation index was 5.1 (in the control group) and 7.9
(in the transfected group) respectively. (p<0.05) These findings
show that GLP-1 stimulated the secretion of insulin under high
glucose conditions but did not stimulate insulin secretion under
low glucose conditions.
Example 6
Delivery of p.beta.GLP1/PAGA Complex to Type 2 Diabetic Animals
[0067] In this example, the PAGA/p.beta.GLP1 complex was delivered
into type 2 diabetic animals in vivo.
[0068] The type 2 diabetic animal was ZDF (Zucker Diabetic Fatty)
rats. These rats have homozygote leptin receptor mutations, so they
seem to have similar manifestations of human type 2 diabetes. The
DNA/carrier complexes were prepared by self assembly. PAGA was
dissolved in distilled water. Before addition of PAGA into the DNA,
the DNA was diluted in 5% glucose solution. Ten times diluted
carrier solution was slowly added into the prepared DNA plasmid and
left for 30 minutes to allow for formation of complexes. PAGA was
injected as a control measure. (DNA/PAGA weight ratio was 1:1.23)
The total volume of DNA/PAGA complex was 2.5 mL. (in 5% glucose
solution)
[0069] Before injection, the rats were incised in their right side
neck areas under anesthesia to expose their jugular veins. The
solution containing p.beta.GLP1/PAGA complex was injected through
the jugular vein. One rat was injected with 250 .mu.g of plasmid
and another was injected with 50 .mu.g of plasmid.
[0070] The blood glucose levels were checked every other day using
a portable glucometer (Accucheck, Roche Inc). The blood glucose
levels were normalized 7 days after injection of the
p.beta.GLP1/PAGA complex. Also, normal glucose levels were
sustained for 4 weeks. The glucose levels of the treatment group
were maintained at less than 150 mg/dL. (FIG. 4.)
Example 7
Delivery of p.beta.GLP1/Biodegradable Polymeric Gene Carrier
Complex to Patients with Type 2 Diabetes
[0071] In this example, the PAGA/p.beta.GLP1 complex will be
delivered into type 2 diabetic patients in vivo.
[0072] The PAGA/p.beta.GLP1 complex prepared as in Example 6 will
be injected into the peripheral vein of patients. Each patient will
be injected with 20 mg of plasmid DNA. (Weight ratio of DNA/PAGA
will be 1:1.23). Total volume of p.beta.GLP1/PAGA complex solution
will be 200 mL, so it will be injected 50 mL bolus I.V. every day
for four days. Same amount of PAGA (24.6 mg in 200 mL, 5% glucose
solution) will be injected to control group. Control and treatment
group will be composed of twenty type 2 diabetics, respectively.
The blood samples will be collected every three days for plasma
insulin and GLP-1 levels. And blood glucose concentration will be
measured four times every day. It is expected that among the
patient who has the treatment of p.beta.GLP1/PAGA complex, the
blood glucose levels should be normalized about five to ten days
after injection of the p.beta.GLP1/PAGA complex. Also, normal
glucose levels is expected to be sustained for at least weeks.
[0073] Thus, among the various embodiments taught there has been
disclosed a composition and method for delivering the GLP-1 gene,
both in vitro and in vivo, for the treatment of type 2 diabetes. It
will be readily apparent to those skilled in the art that various
changes and modifications of an obvious nature may be made without
departing from the spirit of the invention, and all such changes
and modifications are considered to fall within the scope of the
invention as defined by the appended claims.
Sequence CWU 1
1
511201DNAchickenpromoter and enhancer881, 905, 942, 964, 1043n
represents a, t, c, or g. 1agatcttcaa tattggccat tagccatatt
attcattggt tatatagcat 50aaatcaatat tggctattgg ccattgcata cgttgtatct
atatcataat 100atgtacattt atattggctc atgtccaata tgaccgccat
gttggcattg 150attattgact agttattaat agtaatcaat tacggggtca
ttagttcata 200gcccatatat ggagttccgc gttacataac ttacggtaaa
tggcccgcct 250ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
tgacgtatgt 300tcccatagta acgccaatag ggcatttcca ttgacgtcaa
tgggtggagt 350atttacggta aactgcccac ttggcagtac atcaagtgta
tcatatgcca 400agtccgcccc ctattgacgt caatgacggt aaatggcccg
cctggcatta 450tgcccagtac atgaccttac gggactttcc tacttggcag
tacatctacg 500tattagtcat cgctattacc atggtgatgc ggttttggca
gtacaccaag 550ggcgggatag cggtttgact cacggggatt tccaagtctc
caccccattg 600acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
ctttccaaaa 650tgtcgtaata accccgcccc gttgacgcaa atgggcggta
ggcgtgtacg 700gtgggaggtc tatataagca gagctcgttt agtgaaccgt
cagatcacta 750gaagctttat tgcggtagtt tatcacagtt aaattgctaa
cgcagtcagt 800gcttctgaca caacagtctc gaacttaagc tgcagaagtt
ggtcgtgagg 850cactgggcag gtaagtatca aggttacaag ncaggtttaa
ggagaccaat 900agaancttgg gcttgtcgag acagagaaga ctcttgcgtt
tntgataggc 950acctattggt cttnctgaca tccactttgc ctttctctcc
acaggtgtcc 1000actcccagtt caattacagc tcttaggcta gagtacttaa
tangactcac 1050tataggctag cctcgagaat tcacgcgtgg taccatgcgt
caacgtcgtc 1100atgctgaagg gacctttacc agtgatgtaa gttcttattt
ggaaggccaa 1150gctgccaagg aattcattgc ttggctggtg aaaggccgag
gatagtctag 1200a 120124120DNAchickenpromoter and enhancer881, 905,
942, 964, 1043n represents a, t, c, or g. 2agatcttcaa tattggccat
tagccatatt attcattggt tatatagcat 50aaatcaatat tggctattgg ccattgcata
cgttgtatct atatcataat 100atgtacattt atattggctc atgtccaata
tgaccgccat gttggcattg 150attattgact agttattaat agtaatcaat
tacggggtca ttagttcata 200gcccatatat ggagttccgc gttacataac
ttacggtaaa tggcccgcct 250ggctgaccgc ccaacgaccc ccgcccattg
acgtcaataa tgacgtatgt 300tcccatagta acgccaatag ggcatttcca
ttgacgtcaa tgggtggagt 350atttacggta aactgcccac ttggcagtac
atcaagtgta tcatatgcca 400agtccgcccc ctattgacgt caatgacggt
aaatggcccg cctggcatta 450tgcccagtac atgaccttac gggactttcc
tacttggcag tacatctacg 500tattagtcat cgctattacc atggtgatgc
ggttttggca gtacaccaag 550ggcgggatag cggtttgact cacggggatt
tccaagtctc caccccattg 600acgtcaatgg gagtttgttt tggcaccaaa
atcaacggga ctttccaaaa 650tgtcgtaata accccgcccc gttgacgcaa
atgggcggta ggcgtgtacg 700gtgggaggtc tatataagca gagctcgttt
agtgaaccgt cagatcacta 750gaagctttat tgcggtagtt tatcacagtt
aaattgctaa cgcagtcagt 800gcttctgaca caacagtctc gaacttaagc
tgcagaagtt ggtcgtgagg 850cactgggcag gtaagtatca aggttacaag
ncaggtttaa ggagaccaat 900agaancttgg gcttgtcgag acagagaaga
ctcttgcgtt tntgataggc 950acctattggt cttnctgaca tccactttgc
ctttctctcc acaggtgtcc 1000actcccagtt caattacagc tcttaggcta
gagtacttaa tangactcac 1050tataggctag cctcgagaat tcacgcgtgg
taccatgcgt caacgtcgtc 1100atgctgaagg gacctttacc agtgatgtaa
gttcttattt ggaaggccaa 1150gctgccaagg aattcattgc ttggctggtg
aaaggccgag gatagtctag 1200atagagtcga cccgggcggc cgcttcgagc
agacatgata agatacattg 1250atgagtttgg acaaaccaca actagaatgc
agtgaaaaaa atgctttatt 1300tgtgaaattt gtgatgctat tgctttattt
gtaaccatta taagctgcaa 1350taaacaagtt aacaacaaca attgcattca
ttttatgttt caggttcagg 1400gggagatgtg ggaggttttt taaagcaagt
aaaacctcta caaatgtggt 1450aaaatcgata aggatccggg ctggcgtaat
agcgaagagg cccgcaccga 1500tcgcccttcc caacagttgc gcagcctgaa
tggcgaatgg acgcgccctg 1550tagcggcgca ttaagcgcgg cgggtgtggt
ggttacgcgc agcgtgaccg 1600ctacacttgc cagcgcccta gcgcccgctc
ctttcgcttt cttcccttcc 1650tttctcgcca cgttcgccgg ctttccccgt
caagctctaa atcgggggct 1700ccctttaggg ttccgattta gtgctttacg
gcacctcgac cccaaaaaac 1750ttgattaggg tgatggttca cgtagtgggc
catcgccctg atagacggtt 1800tttcgccctt tgacgttgga gtccacgttc
tttaatagtg gactcttgtt 1850ccaaactgga acaacactca accctatctc
ggtctattct tttgatttat 1900aagggatttt gccgatttcg gcctattggt
taaaaaatga gctgatttaa 1950caaaaattta acgcgaattt taacaaaata
ttaacgctta caatttcctg 2000atgcggtatt ttctccttac gcatctgtgc
ggtatttcac accgcatatg 2050gtgcactctc agtacaatct gctctgatgc
cgcatagtta agccagcccc 2100gacacccgcc aacacccgct gacgcgccct
gacgggcttg tctgctcccg 2150gcatccgctt acagacaagc tgtgaccgtc
tccgggagct gcatgtgtca 2200gaggttttca ccgtcatcac cgaaacgcgc
gagacgaaag ggcctcgtga 2250tacgcctatt tttataggtt aatgtcatga
taataatggt ttcttagacg 2300tcaggtggca cttttcgggg aaatgtgcgc
ggaaccccta tttgtttatt 2350tttctaaata cattcaaata tgtatccgct
catgagacaa taaccctgat 2400aaatgcttca ataatattga aaaaggaaga
gtatgagtat tcaacatttc 2450cgtgtcgccc ttattccctt ttttgcggca
ttttgccttc ctgtttttgc 2500tcacccagaa acgctggtga aagtaaaaga
tgctgaagat cagttgggtg 2550cacgagtggg ttacatcgaa ctggatctca
acagcggtaa gatccttgag 2600agttttcgcc ccgaagaacg ttttccaatg
atgagcactt ttaaagttct 2650gctatgtggc gcggtattat cccgtattga
cgccgggcaa gagcaactcg 2700gtcgccgcat acactattct cagaatgact
tggttgagta ctcaccagtc 2750acagaaaagc atcttacgga tggcatgaca
gtaagagaat tatgcagtgc 2800tgccataacc atgagtgata acactgcggc
caacttactt ctgacaacga 2850tcggaggacc gaaggagcta accgcttttt
tgcacaacat gggggatcat 2900gtaactcgcc ttgatcgttg ggaaccggag
ctgaatgaag ccataccaaa 2950cgacgagcgt gacaccacga tgcctgtagc
aatggcaaca acgttgcgca 3000aactattaac tggcgaacta cttactctag
cttcccggca acaattaata 3050gactggatgg aggcggataa agttgcagga
ccacttctgc gctcggccct 3100tccggctggc tggtttattg ctgataaatc
tggagccggt gagcgtgggt 3150ctcgcggtat cattgcagca ctggggccag
atggtaagcc ctcccgtatc 3200gtagttatct acacgacggg gagtcaggca
actatggatg aacgaaatag 3250acagatcgct gagataggtg cctcactgat
taagcattgg taactgtcag 3300accaagttta ctcatatata ctttagattg
atttaaaact tcatttttaa 3350tttaaaagga tctaggtgaa gatccttttt
gataatctca tgaccaaaat 3400cccttaacgt gagttttcgt tccactgagc
gtcagacccc gtagaaaaga 3450tcaaaggatc ttcttgagat cctttttttc
tgcgcgtaat ctgctgcttg 3500caaacaaaaa aaccaccgct accagcggtg
gtttgtttgc cggatcaaga 3550gctaccaact ctttttccga aggtaactgg
cttcagcaga gcgcagatac 3600caaatactgt tcttctagtg tagccgtagt
taggccacca cttcaagaac 3650tctgtagcac cgcctacata cctcgctctg
ctaatcctgt taccagtggc 3700tgctgccagt ggcgataagt cgtgtcttac
cgggttggac tcaagacgat 3750agttaccgga taaggcgcag cggtcgggct
gaacgggggg ttcgtgcaca 3800cagcccagct tggagcgaac gacctacacc
gaactgagat acctacagcg 3850tgagctatga gaaagcgcca cgcttcccga
agggagaaag gcggacaggt 3900atccggtaag cggcagggtc ggaacaggag
agcgcacgag ggagcttcca 3950gggggaaacg cctggtatct ttatagtcct
gtcgggtttc gccacctctg 4000acttgagcgt cgatttttgt gatgctcgtc
aggggggcgg agcctatgga 4050aaaacgccag caacgcggcc tttttacggt
tcctggcctt ttgctggcct 4100tttgctcaca tggctcgaca
4120320DNAArtificial Sequenceprimer_bindPCR primer 3cagaagttgg
tcgtgaggca 20420DNAArtificial Sequenceprimer_bindPCR primer
4gcctttcacc agccaagcaa 20512DNAArtificial Sequenceprotein_bindFurin
cleavage site 5cgtcaacgtc gt 12
* * * * *